Abstract

Biogenesis of the plant secondary cell wall involves many important aspects, such as phenolic compound deposition and often silica encrustation. Previously, we demonstrated the importance of the exocyst subunit EXO70H4 for biogenesis of the trichome secondary cell wall, namely for deposition of the autofluorescent and callose-rich cell wall layer. Here, we reveal that EXO70H4-driven cell wall biogenesis is constitutively active in the mature trichome, but also can be activated elsewhere upon pathogen attack, giving this study a broader significance with an overlap into phytopathology. To address the specificity of EXO70H4 among the EXO70 family, we complemented the exo70H4-1 mutant by 18 different Arabidopsis (Arabidopsis thaliana) EXO70 paralogs subcloned under the EXO70H4 promoter. Only EXO70H4 had the capacity to rescue the exo70H4-1 trichome phenotype. Callose deposition phenotype of exo70H4-1 mutant is caused by impaired secretion of PMR4, a callose synthase responsible for the synthesis of callose in the trichome. PMR4 colocalizes with EXO70H4 on plasma membrane microdomains that do not develop in the exo70H4-1 mutant. Using energy-dispersive x-ray microanalysis, we show that both EXO70H4- and PMR4-dependent callose deposition in the trichome are essential for cell wall silicification.

The exocyst is a protein complex conserved across all eukaryotes, composed of eight subunits with a rod-like shape. Its main function described in yeast is tethering secretory vesicles to the plasma membrane (PM; Munson and Novick, 2006). Two exocyst subunits, SEC3 and EXO70, were described as spatial landmarks for polarized secretion in yeast. It was clearly shown that SEC3 is capable of working as a landmark by itself, whereas EXO70 likely requires additional signaling factors to work (Luo et al., 2014), such as small GTPases (Wu et al., 2010). The rest of the complex, also referred to as the exocyst core, is associated with secretory vesicles and is regulated via RAB GTPase interactions (Robinson et al., 1999). Most of the time, all exocyst subunits form a relatively stable holocomplex in yeast (Heider et al., 2016; Picco et al., 2017). The interaction of the exocyst core with SEC3 and EXO70 subunits at the PM mediates the tethering of the vesicle to the membrane, followed by the SNARE protein-mediated fusion of the vesicle with the membrane (Heider and Munson, 2012; Yue et al., 2017). Besides conventional roles, the animal EXO70 protein was shown to induce membrane curvature (Zhao et al., 2013).

In comparison with yeast and mammalian genomes, which encode only one or two EXO70s, land plant genomes encode a high number of EXO70 paralogs, which likely emerged during land colonization. The Arabidopsis (Arabidopsis thaliana) genome encodes 23 EXO70 paralogs (Elias et al., 2003; Cvrčková et al., 2012). EXO70A1, the most basal EXO70, has been shown to be involved in the secretion of membrane proteins such as PIN1 or BRI1 (Drdová et al., 2013). It also is crucial for the proper development of the Casparian strip (Kalmbach et al., 2017). EXO70A1 also is recruited to the microtubules decorating xylem secondary cell wall thickenings in a COG/VETH-dependent manner, where EXO70A1 recruits the exocyst complex and is responsible for the development of the cell wall thickenings (Oda et al., 2015; Vukašinović et al., 2017). Another example of the exocyst recruitment was shown on EXO70B1, which is recruited to the PM by RIN4 (Sabol et al., 2017). Thus, besides direct lipid interaction, plant EXO70s also seem to be recruited by other proteins. Besides secretion, the EXO70B1 subunit functions in autophagy (Kulich et al., 2013), immune responses (Stegmann et al., 2013; Zhao et al., 2015), and stomatal opening (Hong et al., 2016; Seo et al., 2016). EXO70H1 and EXO70B2 were shown to facilitate the defense papilla buildup (Pecenková et al., 2011). Recently, we showed a role of the EXO70H4 paralog in Arabidopsis trichome cell wall maturation (Kulich et al., 2015).

Arabidopsis leaf trichomes are unicellular outgrowths with very specific polarized shape and two to four (but most frequently three) sharp branches. Their shape is easily and visibly distorted by numerous mutations, which, together with their large size and good accessibility, led to their popularity as a model for studies of plant cell morphogenesis (Hülskamp et al., 1994). They have been especially vital for studies of microtubule- and actin-dependent morphogenetic events (Saedler et al., 2004; Tian et al., 2015), but also serve as a model for deposition of cell wall components (Sinlapadech et al., 2007; Bischoff et al., 2010).

We previously demonstrated that the Arabidopsis trichome cell wall consists of two distinct domains, the basal thin cell wall of the bulb and apical thick cell wall. These domains are separated by a callose-rich structure named the Ortmannian ring (OR). Only the apical cell wall domain consists of an outer and inner layer. We showed that the development of the inner cell wall layer depends on the exocyst subunit EXO70H4 (Kulich et al., 2015). However, we did not provide a detailed description, subcellular localization, and the functional context of EXO70H4. Here, we focused on the EXO70H4-dependent callose deposition, as the lack of callose is one of the most prominent defects of the exo70H4-1 mutant and because callose deposition is of great interest due to its involvement in plant immunity.

In contrast to cellulose, a β-1-4-glucan that is crystallized into permanent microfibrils, callose is deposited as an amorphous plug, and often only transiently, because it is specifically and rapidly degraded by the hydrolytic enzymes β-1-3-glucanases (Levy et al., 2007). Callose is synthesized at the PM by large glucan synthase complexes called callose synthases (CalS) or glucan synthase-like (GSL). The Arabidopsis genome contains 12 CalS genes, which fall into two groups (Verma and Hong, 2001). CalS from the first group contain up to 50 exons and are some of the longest genes found in the Arabidopsis genome. The second group consists of CalS11 (GSL1) and CalS12 (GSL5), containing two and three exons, respectively (Hong et al., 2001). Different CalS are expressed in a tissue-specific way in response to diverse physiological conditions (Dong et al., 2008).

In this study, we worked with two callose synthase proteins: CalS9 (GSL10) and CalS12. CalS9 is known to act in male gametophyte development with mutants defective in pollen mitotic division (Töller et al., 2008; Huang et al., 2009), and it is one of the most transcribed callose synthases in the trichome (Jakoby et al., 2008). CalS12, also known as powdery mildew resistant 4 (PMR4), is a stress-induced callose synthase (Vogel and Somerville, 2000). Knockout mutants lack pathogen-induced callose deposits (Jacobs et al., 2003; Ellinger et al., 2013). One of the possible roles for the callose deposits may be inducing mechanical stiffness in the cell wall by supporting its silicification.

Silica is a nonessential micronutrient absorbed by plants in the form of silicic acid, Si(OH)4, and deposited in different amounts into cell walls of various tissues and structures. Typically, trichome cell walls of many plants are encrusted with silica. Some observations suggest that silica deposition may be related to callose synthesis. The co-occurrence of callose and silica deposition was previously shown in several plant species, including silica hyperaccumulators, for example, in the common horsetail (Equisetum arvense; Law and Exley, 2011), in epidermal trichomes of numerous species (Waterkeyn and Dupont, 1982), Selaginella (Webster, 1992), and Arabidopsis (Brugiére and Exley, 2017). Callose was suggested as an inducing rather than catalyzing element of silicification, operating as a supportive matrix for the specific condensation of silicic acid into silica nanoparticles (Brugiére and Exley, 2017). There also is evidence that carbohydrates other than callose can act as organic matrices for silicification (Perry et al., 1987; Leroux et al., 2013; Guerriero et al., 2016), but also that in some cases no carbohydrates are needed (Hodson, 2016).

Despite this considerable evidence for the dependency of silica deposition on callose synthesis, a comprehensive analysis was still missing. In this report, we demonstrate that callose is indispensable for silica deposition in Arabidopsis trichomes.

RESULTS

EXO70H4 Is Localized to the OR and a PM Domain above It

To observe the localization of the EXO70H4 protein, we generated native promoter-driven constructs with an N-terminal GFP and with mCHERRY (GFP-EXO70H4, mCH-EXO70H4). These constructs were proven to be functional, as they fully complemented the exo70H4-1 mutation described previously (Kulich et al., 2015). In the absence of stress, EXO70H4 constructs were exclusively expressed in the trichome; however, the EXO70H4 promoter also was active in other epidermal cells, showing the capacity of EXO70H4 to be activated elsewhere (Fig. 1A). This also was true for other EXO70 paralogs expressed under the EXO70H4 promoter (see further), suggesting posttranscriptional regulation of EXO70H4. To show whether GFP-EXO70H4 localizes to specific PM domains, we looked for colocalization with the well-established PM marker mCHERRY-PIP1-4 (Geldner et al., 2009; Fig. 1B). Unfortunately, PIP1-4 is almost completely degraded in the mature trichome.

EXO70H4 localization in the trichome. A, EXO70H4 (green) under its native promoter is only visible in the trichome (left), but the EXO70H4 promoter is active also in other epidermal cells (right). Magenta represents chlorophyll; arrowhead points at the OR. B, EXO70H4 colocalizes with PM marker PIP1-4. Graph on the right represents plot of the profile depicted as white dotted line on the left. C, EXO70H4p::mGFP:EXO70H4 (green) localizes to the OR (white arrowheads) and the apical domain above the OR, which produces a highly autofluorescent cell wall (blue). Chlorophyll is in magenta. Left, Single section; middle, single section with transmission; right, z projection. D, A detail of the OR labeled by mCH-EXO70H4 (yellow) and the border of the apical autofluorescent cell wall domain (blue) and the basal domain. Top, Autofluorescence; bottom, autofluorescence and mCH-EXO70H4. E, Detailed view of the mCH-EXO70H4-positive cell wall ingrowths. Top, Transmission; bottom, transmission and mCH-EXO70H4. F, Plasmolysis of a trichome with labeled mCH-EXO70H4, without (top) and with (bottom) developed cell wall ingrowths. The differential attachment of the PM to the cell wall is visible. Bars = 20 μm.

The localization of EXO70H4 very well matches the callose-rich and autofluorescent cell wall shown in Kulich et al. (2015). The XFP-EXO70H4 signal is always present at the OR and above it (apical domain) throughout trichome cell wall development (Fig. 1, A, C, and D). The presence of the EXO70H4 signal was always accompanied by cell wall autofluorescence. The basal PM domain beneath the OR is devoid of GFP-EXO70H4 (Fig. 1, A and D). Identical results were obtained using the mCHERRY construct mCH-EXO70H4 (Fig. 1, D–F). In young trichomes, the signal above the OR is homogeneous. In older trichomes, the signal becomes more speckled and accumulates at the differential interference contrast-visible cell wall ingrowths, which are a common feature of older trichomes (Fig. 1E). Upon plasmolysis, the mCH-EXO70H4 signal is easily separated from the cell wall of the young trichomes but is attached to the cell wall that has developed ingrowths along cytoplasmic strands (Fig. 1F). To confirm that these are actual cell wall ingrowths, we investigated the trichome cell wall from the inside using environmental scanning electron microscopy (ESEM; Supplemental Fig. S1).

On these ingrowths, mCH-EXO70H4 transiently colocalizes with the core exocyst subunits GFP-SEC8 and EXO84-GFP expressed under their respective natural promoters (Fig. 2). The core exocyst subunit signal is, however, visible also in the cytoplasm and in other membrane domains, suggesting their general function in secretion. These data fit with our previous yeast two-hybrid study, where EXO70H4 also physically interacted with the Arabidopsis exocyst subunits SEC5A, SEC6, and EXO84b (Kulich et al., 2015).

Colocalization of the core exocyst subunits with EXO70H4. SEC8 and EXO84 under their own natural promoters largely localize to the cytoplasm and partially localize to the EXO70H4-positive compartments. The ratio of both signals changes over time. Bars = 20 μm.

Flg22 Induces EXO70H4 Expression in Epidermal Pavement Cells

Because the composition of the trichome inner cell wall layer highly resembles the pathogen-induced cell wall appositions, we investigated whether there is a possible function of EXO70H4 beyond the trichome. As shown in Figure 1A, the EXO70H4 promoter has a capacity to drive EXO70H4 expression in other epidermal cells, but the EXO70H4 protein is absent. Public microarray data suggest an elevation of the EXO70H4 mRNA signal upon flg22 treatment. Therefore, we applied 1 μm flg22 by spraying on the mature Arabidopsis rosettes (24 d old). Four hours after the treatment, the first visible signal appeared in the epidermal pavement cells and peaked approximately 5 h after induction (Fig. 3A). The signal disappeared again 12 h after induction. We got similar results with both GFP and mCHERRY lines and quantified the fluorescence intensity (Fig. 3B). These results are supported by the western blot analysis of the total cell extract using an anti-GFP antibody and document similar up-regulation using chitin as elicitor (Fig. 3C). In many cells, the EXO70H4 signal was not evenly distributed across the cell surface and formed small domains of higher signal intensity. These domains developed cell wall autofluorescence similar to the autofluorescence of the trichome apical cell wall (Fig. 3D).

EXO70H4 up-regulation by flg22 in the leaf pavement cells. A, Representative images of leaves 5 h after flg22 treatment (spraying by 1 μm flg22 in 0.05% Silwet and 0.05% Silwet as control). Yellow, mCH-EXO70H4; magenta, chlorophyll; grays, transmission. Arrowheads depict domains with enchiched EXO70H4 signal. Scale bar = 20 μm. B, Quantification of the EXO70H4 signal intensity out of 10 plants and 100 cells. Similar results were obtained in three independent replicates, with both GFP- and mCHERRY-labeled constructs. C, Up-regulation of EXO70H4 in total cell extract from whole rosettes of GFP-EXO70H4 plants, 5 h after flg22 and chitin treatment. The white line is where one empty lane was left on the gel due to overflow. SEC6 was used as a loading control. D, GFP-EXO70H4 (green) forms a microdomain with enriched signal. This is accompanied by development of cell wall autofluorescence (blue). The arrow shows the position of the plot profile in D. Scale bar = 5 μm. Right, Plot profile demonstrating spatial separation of EXO70H4 and cell wall autofluorescence.

EXO70H4 Differs from Other Arabidopsis EXO70 Paralogs in Its Function and Subcellular Localization

To learn more about the specificity of EXO70H4 function, we performed a cross-complementation analysis, in which we subcloned multiple EXO70 paralogs under the EXO70H4 promoter. We subcloned 18 different EXO70 paralogs in the same fashion under EXO70H4 promoter (EXO70H4p::GFP:EXO70XY). We selected at least one gene from each subfamily (A–G) and all members of the subfamily H. Then, we transformed these constructs into the exo70H4-1 mutant background and observed the development of trichome autofluorescence (Fig. 4, A and B) and trichome callose deposition (Supplemental Fig. S2). Both of these parameters provided us with identical results. Apart from EXO70H4, no other paralog could restore either the callose or autofluorescence, suggesting that the function of EXO70H4 is highly specific.

Most EXO70 paralogs showed no PM localization in the trichome (Supplemental Fig. S3). While EXO70A1 was uniformly distributed over the whole PM of the trichome (even beneath the OR; Fig. 4C), EXO70B1 and most of the other EXO70 paralogs showed cytoplasmic and nuclear localization.

Despite using an identical experimental setup, some of the EXO70 proteins (H1, E2, G2, H2, H3, H5, and H6) were not detected in the trichome, and therefore, we cannot exclude that some of these have the capacity to complement EXO70H4 on the protein level (Supplemental Fig. S3). This may be due to microRNA (miRNA) regulation of the EXO70 mRNA. We identified several miRNAs that may interfere with EXO70. The number of predicted interfering RNAs (based on Yi et al. [2015] database) is enhanced in the EXO70H subfamily (Supplemental Table S1).

EXO70H4 Is Essential for PM Localization of Callose Synthases in the Trichome

Since we showed that callose is absent from the exo70H4-1 mutant trichomes (Kulich et al., 2015), we further investigated the callose synthase delivery to the PM. We worked with two callose synthases and generated ubiquitin promoter-driven callose synthase constructs UBQ::GFP:PMR4 (GFP-PMR4) and UBQ::GFP:CALS9 (GFP-CALS9). Here, we focus on PMR4, which is essential for the callose production in the trichome. We obtained similar results using CALS9. These can be found in the supplements (Supplemental Fig. S4).

Next, we introduced UBQ::GFP:PMR4 into the wild type and the exo70H4-1 mutant line. In the wild-type trichome, the signal of both callose synthases was visible in immobile membrane speckles at the PM and also on mobile membrane bodies, possibly representing multiple steps of the secretory pathway (Fig. 6A). To separate mobile PMR4 fraction from the immobile PM dots, we performed time-series imaging and minimal-intensity projections (Fig. 6B). In the exo70H4-1 mutant plants, the signal from the PM speckles was lost, suggesting a secretory defect of both of these callose synthases (Fig. 6, B–D).

EXO70H4 recruits PMR4 to the ingrowths of the trichome cell wall. A, Left, Overall view of the trichome expressing mCH-EXO70H4 (magenta) and GFP-PMR4 (green). Projection of 14 sections. Yellow dotted square depicts detailed view on the right. Right, Detailed view of a single section with cell wall ingrowths decorated by EXO70H4 and PMR4 with a 2D histogram for the colocalization (Pearson’s R value, 0.35; Li’s ICQ value, 0,127). B, PMR4-positive cell wall ingrowths are absent in the exo70H4-1 mutant. In the single frame, both the mobile endomembrane fraction and the immobile ingrowths are visible. The mobile fraction is reduced by minimal intensity projection (min. proj.). C, A detail of the ingrowths from B. D, Quantification of the immobile dots in a blind study using the minimal intensity projections. WT, Wild type; bars = 20 μm.

By colocalization of EXO70H4p::mCHERRY:EXO70H4 with UBQ::GFP:PMR4, we show that the callose synthase speckles also are EXO70H4 positive (Fig. 6A). As described above, the signal of callose synthases was first visible in smaller transient speckles on the PM of young trichomes and developed later into large speckles. Taken together, our data show the dependency of callose synthase secretion on the EXO70H4 protein.

Silica Accumulation Is Dependent on Callose Deposition and Thus on the EXO70H4-Dependent PMR4 Secretion

While doing our ESEM studies of untreated biological samples (Tihlaříková et al., 2013; Neděla et al., 2015), we applied energy-dispersive x-ray spectroscopy for a semiquantitative analysis of elements in the trichome. Surprisingly, apart from heavy metals, we noticed a significant amount of silica in the domain above the OR. This silica encrustation was absent in exo70H4-1 mutant trichomes. To determine whether this was a direct effect of the exo70H4-1 mutation, we next included a pmr4 mutant (which lacks callose in the trichome), and we increased the amount of silica in the soil by watering with sodium silicate solution (final concentration 2 mm). As shown in Figure 7, both the pmr4 and the exo70H4-1 mutants had dramatically reduced silica levels in the trichomes. Therefore, we conclude that callose deposition is essential for cell wall silicification and that the exo70H4-1 silica phenotype is a secondary phenotype, contingent on the impaired callose synthase delivery and the subsequent absence of callose synthesis.

DISCUSSION

In our previous study, we demonstrated the EXO70H4-dependent development of the callose-rich cell wall in the Arabidopsis trichome. Here, we show that EXO70H4 acts by promoting the secretion of callose synthase. Surprisingly, no other EXO70 subcloned under the EXO70H4 promoter was able to complement the exo70H4-1 phenotype, despite high sequence similarity of some paralogs (Cvrčková et al., 2012). Since EXO70 proteins in general act as spatial landmarks for secretion, it is likely that the specificity of EXO70 paralogs reflects their differential target binding capacities. EXO70H4 decorates a specific PM subdomain in the trichome, while the most ancestral EXO70A1 localizes all over the trichome PM. This is consistent with the previous model of multiple recycling domains within one cell (Zárský et al., 2009). We also observed that some of the EXO70 paralogs with cytoplasmic localization in the trichome localize to the PM in the pavement cells (e.g. EXO70B1), suggesting differential regulatory mechanisms.

The cross-complementation analysis suggests that the EXO70H4-positive trichome PM domains have a highly distinctive character and cannot be recognized by other EXO70 paralogs. Previously, it was proposed that the EXO70 paralogs differ just in their expression pattern (Li et al., 2010). In this study, we demonstrate that there is functional divergence between the paralogs. This also is supported by other studies showing specific EXO70 roles (Kulich et al., 2013; Zhao et al., 2015) and recently by the specific PM domain localizations of NtEXO70A1 and NtEXO70B1 in tobacco (Nicotiana tabacum) pollen tubes (Sekereš et al., 2017).

Since EXO70 proteins are putative landmarks for secretion, the specificity of EXO70 paralogs could mainly be determined by different localization signals. For example, during xylogenesis, EXO70A1 localization to cortical microtubules is maintained by COG-VETH proteins (Oda et al., 2015; Vukašinović et al., 2017). Also, as we showed recently, EXO70B1 PM localization can be achieved by protein-protein interaction with NOI family proteins (Sabol et al., 2017). We speculate that a similar mechanism, but with different proteins, may be responsible for the specific localization of many EXO70 paralogs, causing their functional diversity. Whether the process of EXO70H4 localization is mediated by specific lipid-binding properties or by interacting proteins is the subject of our follow-up study.

The initially homogenous PM signal of GFP-EXO70H4 develops later into stable speckles, which then form ingrowths of the cell wall. Similar behavior of the exocyst subunits was observed during xylogenesis, where EXO70A1-tagRFP first localized dispersedly to the PM and later on gradually organized into a bundled pattern (Vukašinović et al., 2017). Such stabilization of the polarity was observed previously in budding yeast (Saccharomyces cerevisiae; Brennwald and Rossi, 2007). The mechanism of this stabilization in plants is not yet known, but in our opinion it may be achieved by a positive feedback loop, whereby the original EXO70 attracts vesicles with more exocyst subunits.

Unfortunately, not all the EXO70 constructs in our cross-complementation study were expressed in the trichome. We explain this by a predicted RNA interference regulation, which is common among stress-induced transcripts (Sunkar and Zhu, 2004), or by ubiquitination, which was previously manifested as a possible step in EXO70 protein regulation (Samuel et al., 2009; Stegmann et al., 2012; Seo et al., 2016). Very likely, EXO70s are subjected to a high degree of regulation at multiple levels.

Here, we show a secretory defect of the exo70H4-1 mutation on one type of cargo: two callose synthases, which both localized and behaved similarly despite their functional classification. Of these, only CALS12 was biologically relevant for callose synthesis in the trichome. More cargo affected by the exo70H4-1 mutation must exist, since the trichomes lacking only callose and silica in the case of the pmr4 mutation are still mechanically relatively stiff and accumulate autofluorescent compounds and metals, unlike exo70H4-1 mutants.

The inner cell wall of the trichome shares many similarities with the pathogen-induced cell wall (being rich in callose, silica, and phenolic compounds; Russo and Bushnell, 1989; Ghanmi et al., 2004), and as we show here, EXO70H4 has the capacity to contribute to such a cell wall biogenesis, since the EXO70H4 protein appears in nontrichome cells upon bacterial elicitor treatment, allowing PMR4 and other cargo secretion and callose synthesis. Deposited callose thereafter acts as a matrix for silica accumulation, which is known to modulate physical properties of the cell wall and acts as an important line of defense against fungal pathogens (Ghanmi et al., 2004; Fauteux et al., 2006; Vivancos et al., 2015). The exact relationship between callose and the silica accumulation was enigmatic for a long time, although it was clear that these processes are related (Law and Exley, 2011; Exley, 2015). First evidence that callose may be essential for silica deposition was provided recently (Brugiére and Exley, 2017), using chemical staining of silica. In our study, we extend these observations with quantitative and statistically processed data. We also show that callose is not essential for the accumulation of phenolic compounds in the cell wall in contrast to some observations that lignification precedes silicification (Zhang et al., 2013).

While Arabidopsis trichomes contain relatively little silica, trichomes of species such as nettle (Urtica dioica) are well known for their silicified cell wall (Sangster and Hodson, 2007). Cucumber (Cucumis sativus) trichomes also contain silica, and supplementing plants with silica leads to physically stiffer trichomes (Samuels, 1993). In cucumber, basal cells of the trichomes (sometimes referred to as cells surrounding the trichome or the trichome spine) were the site of maximal silica deposition (Samuels et al., 1991a, 1991b; Chérif et al., 1992). Transcriptomic analyses have revealed that wild-type cucumber contains 406-fold more CsEXO70H4 transcript than the trichome-less tbh mutant (Chen et al., 2014). This suggests that the mechanisms we describe in the Arabidopsis trichome may have more general implications for eurosids.

MATERIALS AND METHODS

Plant Material and Growth

If not indicated otherwise, plants were grown in standard growth chamber conditions (long day 16 h:8 h, 100 μm photosynthetically active radiation m−2 s−1). LT 36W/958 T8 BIOVITAL NARVA fluorescent tubes were used. These contain a UV-B peak. All plants were grown in Jiffy soil pellets. The exo70H4-1 mutant was described previously (Kulich et al., 2015), as well as the pmr4-1 mutant (Vogel and Somerville, 2000). As a control, a wild-type sibling of the exo70H4-1 mutant was used. As a control for the exo70H4-1 × rdr6-12 double mutant, rdr6-12 (Peragine et al., 2004) was used.

Callose Staining and Autofluorescence Visualization

To stain for callose, whole leaves were washed for 3 h in acetic acid:ethanol (1:3) solution, washed three times in deionized water, and incubated overnight in aniline blue solution (150 mm KH2PO4 and 0.01% [w/v] aniline blue, pH 9.5). Trichomes were then imaged on leaves or brushed off and imaged.

For autofluorescence development, leaves were placed in between two microscopy slides and dried up overnight. Fifth and sixth leaves of the 24- to 28-d-old rosettes were used for the aniline blue staining and the third youngest visible leaf for autofluorescence observations.

PAMP Treatments

Flg22 (1 μm) or chitosan solution (150 mg/L) with 0.05% Silwet Star wetting agent was sprayed onto the 24-d-old Arabidopsis (Arabidopsis thaliana) rosettes stably transformed with XFP-EXO70H4. Silwet Star (0.05%) was used as a control. Signal was observed 4 to 5 h after the treatment. This experiment was done in triplicate. Spraying was critical factor for the EXO70H4 up-regulation. Protein extract from whole rosettes was used for the western blot analysis, using primary anti-GFP and anti-SEC6 antibodies (Agrisera; AS15 2987 and AS13 2686, respectively).

Construct and Transgenic Line Preparation

For the cloning of all EXO70 paralogs, a multisite gateway approach was used. The EXO70H4 promoter (1 kb upstream) was subcloned into pDONORP4-P1r. GFP in pEN-L1-F-L2 (GFP) was obtained from Karimi et al. (2007). pEN-L1-mCherry-L2 was obtained from Mylle et al. (2013); however, since this construct had a stop codon, we used it as a template to generate a new pDONOR 221-mCherry construct. EXO70 coding sequences were amplified in one or two steps (with att extension primers) and subcloned into the pDONOR P2R-P3 using Gateway BP clonase (Invitrogen). Then, multisite reactions were performed using the EXO70H4 promoter, GFP, EXO70 coding sequence, and destination vector pB7m34GW (BASTA plant selection; Karimi et al., 2007). pDONOR vectors were sequenced using M13 primers. The destination binary constructs were sequenced using M13 primers and two GFP primers (see primer list in Supplemental Table S2). To clone callose synthase PMR4, the genomic fragment was subcloned into pDONOR 221 using Gateway BP clonase and then transferred into the pUBN-GFP vector (Grefen et al., 2010) using Gateway LR clonase. Since the CalS9 genomic fragment is too long to amplify, we isolated the cDNA. This was stitched together from two parts, as none of transcripts had all 42 introns properly spliced. GFP-SEC8 and EXO84b-GFP lines were described previously (Fendrych et al., 2013). All primers in this study are listed in Supplemental Table S2.

All prepared constructs were electroporated into Agrobacterium tumefaciens GV3101 competent cells. The floral dip method (Clough and Bent, 1998) of plant transformation was used, and the transformants were selected on soil by spraying with BASTA (150 mg/L of glufosinate-NH4). At least five individual transformants were observed in each experiment, and at least two biological replicates were made.

ESEM and Energy-Dispersive X-Ray Microanalysis

For a semiquantitative energy-dispersive x-ray microanalysis (EDS), four trichomes from eight leaves were selected for each of the three samples (wild type, pmr4, and exo70H4-1), air dried, and placed on a carbon pad. To maximize detection efficiency and accuracy of the analysis, trichomes were selected according to their shape and shadowing in x-ray maps using a solid-state detector of backscattered electrons and fast EDS mapping. Dried, chemical treatment-free, and conductive coating-free samples were imaged and analyzed using ESEM Quanta 650 FEG equipped with EDS silicon drift detector Bruker Quantax 400 XFlash 6/60 under beam energy 10 keV, beam current 100 pA, working distance 10 mm, and water vapor pressure 100 Pa. The ratio of silicon in the sample was calculated as the median values obtained from all trichomes for each sample.

The cell wall of the mature Arabidopsis trichome was in situ opened using two Kleindiek micromanipulators MM3A-EM, thus without any manipulation, cutting, or contamination outside the specimen chamber of ESEM. The inner surface of the trichome (Supplemental Fig. S1) was imaged using gaseous secondary electron detector under beam energy 10 keV, beam current 50 pA, working distance 11.5 mm, and water vapor pressure 170 Pa.

Acknowledgments

We thank Lukáš Fischer for the motivation to look for the putative EXO70s regulating miRNAs and our technician Marta Čadyová, Patrick Moxon, and Matouš Glanc for a significant improvement of the cloning methods and for constructs provided.

The author responsible for distribution of materials integral to the findings presented in this article in accordance with the policy described in the Instructions for Authors (www.plantphysiol.org) is: Ivan Kulich (kulich{at}natur.cuni.cz).

↵1 This work was supported by the Grant Agency of Czech Republic (grant nos. 14-27329P and GF16-34887L), the Czech Ministry of Education (grant no. NPUI LO1417), and the Grant Agency of Charles University [grant no. GA UK(CZ) 387515]. Microscopy was performed in the Laboratory of Confocal and Fluorescence Microscopy cofinanced by the European Regional Development Fund and the state budget of the Czech Republic (project nos. CZ.1.05/4.1.00/16.0347 and CZ.2.16/3.1.00/21515).